Abstract: Transgenic and gene-editing technologies are essential for gene functional analysis and crop improvement. However, the pleiotropic effects and unknown mechanisms of morphogenic genes have hindered their broader application. In this study, we employed the one-step de novo shoot organogenesis (DNSO) method, and demonstrated that overexpression of the morphogenic gene Arabidopsis thanalia GROWTH-REGULATING FACTOR 5 (AtGRF5) significantly enhanced genetic transformation efficiency in cucurbit crops by promoting callus proliferation and increasing dense cells during regeneration. High-resolution time-series transcriptomics and single-cell RNA sequencing revealed that AtGRF5 overexpression induced auxin-related genes and expanded stem cell populations during cucumber DNSO. Using DNA-affinity purification sequencing (DAP-seq) in combination with spatiotemporal differential gene expression analysis, we identified CsIAA19 as a key downstream target of AtGRF5, with its modulation playing a pivotal role in regeneration. Rescuing CsIAA19 in AtGRF5-overexpressing explant reversed the enhanced callus proliferation and regeneration. To address growth defects caused by AtGRF5 overexpression, we developed an abscisic acid-inducible AtGRF5 expression system, significantly improving transformation and gene-editing efficiency across diverse genotypes while minimizing pleiotropic effects. In summary, this research provides mechanistic insights into AtGRF5-mediated transformation and offers a practical solution to overcome challenges in cucurbit crop genetic modification.

Key words: auxin, cucurbit crops, genetic transformation, GRF, regeneration, scRNA‐Seq